(a) Field of the Invention
The invention relates to biosensors in which a monomolecular biological conjugate layer is attached to a transducing device.
(b) Description of Prior Art
Biosensors are a rapidly emerging technology for detecting and/or measuring the occurrence of biological phenomena or the presence of biological molecules or organisms. A biosensor is any analytical device incorporating a biological material, a biologically derived material or biomimetic intimately associated with or integrated within a physiochemical transducer or transducing microsystem, which may be optical, electrochemical, thermoelectric, piezoelectric or magnetic (Fishman et al., Annu. Rev. Biophys. Biomol. Struct., 27:165-198, 1998). Typical biosensors are formed by attaching a biological molecule, such as an enzyme or an antibody, to a transducer. The biosensor is exposed to an environment in which it is desired to detect bioactivity or a specific biological entity, and signals emitted by the transducer reflect the involvement of the biological molecule in a bioreaction or biointeraction.
Examples of biosensor technology are disclosed in Ahluwalia et al., xe2x80x9cA comparative study of protein immobilization techniques for optical immunosensors,xe2x80x9d Biosensors and Bioelectronics, 7:207-214(1991) and Geddes et al., xe2x80x9cImmobilisation of IgG onto gold surfaces and its interaction with anti-IgG studied by surface plasmon resonance,xe2x80x9d Journal of Immunological Methods, 175:149-60 (1994).
An important issue in the construction of biosensors is the attachment and immobilization of a biological material in relation to the transducer. If the biological molecule is not attached in proper relation to the transducer or in sufficient amount, then the sensor may not operate satisfactorily. Problems encountered with prior biosensor constructions include maintaining sufficient accessibility, density and/or orientation of the biologically active molecule or organism used in the biosensor. Some sensor designs enclose the bioactive molecule in a matrix or membrane. Such designs tend to restrict the accessibility of the bioactive molecule to the moiety which it is intended to interact with and sense and thereby limit the sensitivity of the resulting sensor. Attempts have also been made to directly attach bioactive molecules to sensors and/or substrates. However, such techniques do not necessarily result in optimum density of the bioactive sensor molecules or in a uniform orientation of the bioactive sensor molecule with the active site or epitope in a properly exposed position for effective interaction with the intended moiety. None of the prior art biosensors is provided with a chemical coating which would promote optimum biochemical interactions reactions at the biosensor/test medium interface, thereby enhancing the sensitivity and usefulness of the biosensor. Consequently, despite the efforts of the prior art, there remains a substantial need for improved biosensor designs.
In various fields, attempts have been made to affix molecules on the surfaces of articles or materials to modify their surface properties. For example, Sukenik, C. N. et al. (J. Biomed. Materials Res., 24:1307-1323, 1990) describes the modulation of cell adhesion by modification of titanium surfaces with covalently attached self-assembled monolayers. Wieserman et al., U.S. Pat. No. 4,788,176 teaches a process for chemically bonding a monomolecular layer of phosphorous- containing material to metal oxide/hydroxide particles to form an active material suitable for use as an absorbent. Rhee et al., U.S. Pat. No. 5,328,955 discloses covalent attachment of collagen to organic polymers and their use with implants. However, none of these documents discloses or suggests the use of bioactive conjugates which include covalently attached biologically active molecules to a biosensor surface in order to enhance the sensitivity and/or usefulness of a biosensor.
Advances in the immobilization of affinity ligands and innovation in the merging field of bioelectronics have combined to produce revolutionary new detection devices. Affinity electrodes and biosensors are based on the specific interaction between receptors, enzymes, or antibody molecules and their specific target analytes. The application of this technology to measurement systems has created novel analytical detection devices for such diverse fields as diagnostics, therapeutics, process control, waste and environmental monitoring, computer technology, and the kinetic analysis of the interaction of various biological substances.
Common to every device is a support material to which an immobilized affinity ligand is attached. This ligand may be an enzyme that is designed to monitor the presence of its specific substrate in solution. It may be an antibody that can measure its complementary antigen, or an antigen to detect specific antibodies. The affinity ligand may also be any biospecific molecule that interacts with a particular receptor protein (or vice versa). It can even be an immobilized intact living organism (cellular) that can act on specific substances in the solutions with which it comes in contact.
To detect the interaction of these affinity pairs, a functional biosensor needs an electronic transducer that senses the subtle chemical changes that take place between the immobilized ligand and the specific analyte. The detection process may involve the monitoring of electrical effects such as potentiometric changes, amperometric fluctuations, or capacitance differences; optical effects such as light absorption, scattering, or refractive index; changes in density or mass; acoustical effects such as changes amplitude, frequency, or phase of a sound wave; or thermal differences using sensitive calorimeters. The electronic detector then sends its signals an amplification device that also may process and compute the concentration of the analyte in solution. The output and control of these instruments may be as simple as reading a needle gauge on a device such as a pH meter as complex as sophisticated computerized instruments with programmable interfaces.
The principles of biosensor operation have been employed for decades with oscilloscopes designed to monitor slight changes in electrical phenomena. For instance, an olfactory organ such as the antenna of a butterfly can be placed between the input leads of an oscilloscope and used to detect the interaction of olfactory receptors with various volatile substances. In this case, the initial amplifiers of the receptor-ligand interaction are the olfactory nerves that generate action potentials along their length in response to the binding of specific substances. Important qualitative information can be obtained in such a system, but is of little quantitative use.
In modern biosensor design, a synthetic receptor-ligand surface is constructed that has specificity for a single substance. Since the surface is monospecific and the response varies in proportion to the quantity of ligand in the sample solution, quantitative analytical measurements are possible.
The goal is to form a local concentration of the affinity ligand across the biospecific surface. Correct orientation and retention of activity are important in this process, especially for ligands containing active sites that must interact with specific analytes after immobilization.
In general, entrapment or absorption procedures do not yield stable affinity systems for biosensor design. Such sensors may work for brief periods in the laboratory, but the weak bonds created by noncovalent attachment usually cause severe leakage of the biomolecule off the surface and degradation of performance with use. Entrapment, however does provide a viable immobilization means when attaching cellular ligands to a surface, since the cells are typically surrounded by a polymerized or gelatinous membrane and are unable to break free.
The aim of the present invention is to provide biosensors in which a monomolecular biological conjugate layer is covalently attached either directly to a transducing device or to a metal coating on at least one surface of a transducing device.
In accordance with the present invention there is provided for use a monomolecular biological conjugate layer adapted to attach to a metallic transducing device directly or to a transducing device which has a thin coat of metal. The monomolecular biological conjugate layer has the following structural formula I:
xe2x80x94Rxe2x80x94Xxe2x80x94Pxe2x80x83xe2x80x83I
wherein,
R is O or S, adapted to be covalently attached to a metallic surface;
X is selected from a bond, linear or branched chains of 1 to 30 covalently attached atoms selected from the group consisting of C, N, O, Si or S or other linking atoms, rings of 1 to 20 covalently attached atoms selected from the group consisting of C, N, O, Si or S or other linking atoms and a combination of rings and chains of similar composition; and
P is a covalently-attached biological molecule.
More particularly, in accordance with the present invention, X is selected from one of the following possibilities: a direct bond to a biological molecule; a linear alkyl C1-C30 chain, terminated by COOH, NH2, OH, SH or other functional groups chosen to permit covalent linking to a biological molecule; a linear chain consisting of 1-20 atoms of C interspersed with 1-10 atoms of N, O or S, terminated by COOH, NH2, OH, SH or other functional groups chosen to permit covalent linking to a biological molecule; a linear alkylsilyl SiC1-SiC30 chain, terminated by COOH, NH2, OH, SH or other functional groups chosen to permit covalent linking to a biological molecule; or rings composed of C and/or N, connected directly to a biological molecule or connected by means of linear chains of C, N, O or S atoms, terminated by COOH, NH2, OH, SH or other functional groups chosen to permit covalent linking to a biological molecule.
The X moiety of the biological conjugate is selected depending on the desired P molecule which is to be attached to a transducing device and also is chosen according to the desired spacing distance of the P molecule from the transducing device. Consequently, this leads to the correct orientation of the biological molecule for sensing and/or measuring purposes.
The preferred X moieties in accordance with the present invention are C2-C12 alkyl, which may be substituted or non-substituted, SiC3-SiC12, which may be substituted or non-substituted, and 1,3,5-triazine (cyclic C3N3), which may be substituted or non-substituted.
The X moiety may be substituted with a substituent selected from the group consisting of COOH, NH2, OH, SH, Cl or other groups chosen to permit covalent linking to a biological molecule.
The stably attached biological molecule P includes, but is not limited to, a biological material (such as tissue, microorganisms, whole cells, enzymes, receptors, antibodies or nucleic acids), a biologically derived material or biomimetic. Examples of suitable biological materials include osteopontin, derivatized osteopontin, anti-osteopontin antibodies, bone sialoprotein, bone acidic glycoprotein-75, osteocalcin, osteonectin, bone morphogenetic proteins, transforming growth factors, laminin, type IV collagen, type VIII collagen, enamel proteins (amelogenins and non-amelogenins), anti-amelogenin antibodies, xcex12HS-glycoprotein, fibronectin, cell adhesion peptides, prostaglandin, serum proteins, glucocorticosteroids (dexamethasone), phosphate, phosphoserine, pyrophosphates, phosphothreonine, phosvitin, phosphophoryn, biphosphonates, phosphonates, phosphatases, sulfonates, sulfates, carboxylates, bone and epithelial proteoglycans, mineral and cell binding peptide sequences such as Arginine-Glycine-Aspartic acid (Arg-Gly-Asp), polyaspartate, and other biological molecules capable of interacting with a biological moiety to be sensed or measured.
In accordance with the present invention there is provided a monomolecular biological conjugate layer adapted to covalently attach to a transducing device of a biosensor.
The biosensor can also be coupled with a chemical separations, for example a chromatographic separation process.
Further, the monomolecular biological conjugate layer of the present invention may form a self-assembling monolayer on the transducing device surface.
In addition to covalently attaching the biological molecule, the monomolecular biological conjugate layer inhibits the contamination of the metal surface of the transducing device.
In accordance with the present invention the expression xe2x80x9cmetal coatxe2x80x9d is intended to mean any transducing device material made of solid metal or of a metal sheet or foil, or a material having at least one side or one surface coated with metal.
More particularly, in accordance with the present invention, the metal surface of the transducer may be any metal to which a suitable oxygen or sulfur atom containing functional group can be covalently attached. If the biosensor is to be used in conjunction with a living organism, for example implanted into a living patient, the metal surface of the transducer should be formed of a medically acceptable metallic material. Suitable metals are known to persons skilled in the art. Examples of suitable metals include titanium, stainless steel, tantalum, Vitallium(trademark), gold, silver, platinum and/or alloys thereof. Titanium is the preferred metallic material.
The modified metal surfaces, as in the present invention, may be used for the micro or nanofabrication of molecular patterns or arrays of molecules, which are herein referred to as xe2x80x9cmolecular integrated circuitxe2x80x9d. This form of molecular integrated circuits may have applications in fields such as bioelectronics and molecular electronics.
Another application for the monomolecular biological conjugate layer of the present invention would be in the construct of tips for atomic force microscopy (AFM) and/or for scanning probe microscopy. This would allow the studying of the force or interaction between molecules, where one is attach to a substrate and the other is attached to the tip of AFM.